Institute Biology Leiden (IBL), Leiden University, Leiden, The Netherlands.
FEMS Microbiol Lett. 2010 Apr;305(1):81-90. doi: 10.1111/j.1574-6968.2010.01916.x. Epub 2010 Jan 28.
Live-cell imaging techniques are essential to gain a better understanding of microbial functioning in natural systems, for example in biofilms. Autofluorescent proteins, such as the green fluorescent protein (GFP) and the red fluorescent protein (DsRed), are valuable tools for studying microbial communities in their natural environment. Because of the functional limitations of DsRed such as slow maturation and low photostability, new and improved variants were created such as mCherry. In this study, we developed genetic tools for labeling Gram-negative bacteria in order to visualize them in vitro and in their natural environment without the necessity of antibiotic pressure for maintenance. mcherry was cloned into two broad host-range cloning vectors and a pBK-miniTn7 transposon under the constitutive expression of the tac promoter. The applicability of the different constructs was shown in Escherichia coli, various Pseudomonas spp. and Edwardsiella tarda. The expression of mcherry was qualitatively analyzed by fluorescence microscopy and quantified by fluorometry. The suitability of the constructs for visualizing microbial communities was shown for biofilms formed on glass and tomato roots. In addition, it is shown that mCherry in combination with GFP is a suitable marker for studying mixed microbial communities.
活细胞成像技术对于更好地了解自然系统中的微生物功能至关重要，例如在生物膜中。自发荧光蛋白，如绿色荧光蛋白（GFP）和红色荧光蛋白（DsRed），是研究自然环境中微生物群落的有用工具。由于 DsRed 的功能限制，如成熟缓慢和光稳定性低，因此创建了新的和改进的变体，如 mCherry。在这项研究中，我们开发了用于标记革兰氏阴性细菌的遗传工具，以便在体外和自然环境中对其进行可视化，而无需维持抗生素压力。mcherry 被克隆到两个广谱宿主范围克隆载体和一个 pBK-miniTn7 转座子中，在 tac 启动子的组成型表达下。不同构建体的适用性在大肠杆菌、各种假单胞菌和迟缓爱德华菌中得到了证明。通过荧光显微镜对 mcherry 的表达进行了定性分析，并通过荧光计进行了定量分析。构建体用于可视化微生物群落的适用性在玻璃和番茄根上形成的生物膜上得到了证明。此外，还表明 mCherry 与 GFP 结合是研究混合微生物群落的合适标记物。
